The dynamic process of membrane trafficking within eukaryotic cells involves numerous specialized protein complexes and lipid domains. One particularly intriguing set of proteins is the dynamin family of mechanochemical enzymes; a family of large GTPases potentially involved in nearly all cellular membrane stabilization and fission events. We are interested in examining the dynamic structural properties of these proteins, derived from their mechanochemical properties, and correlate them to their cellular function. Dynamin itself is essential for receptor mediated endocytosis, caveolae internalization and trafficking to and from the Golgi. Dynamin was first implicated in endocytosis when it was discovered to be the mammalian homologue to the shibire gene product in Drosophila. Since then human dynamin mutants overexpressed in mammalian cells were found to effectively block clathrin-mediated endocytosis. We have previously shown that purified dynamin readily assembles into rings and spirals, and others have demonstrated that treatment of synaptosomes with GTPgS induced the formation of dynamin-coated invaginations. This evidence supports the hypothesis that dynamin assembles around the necks of clathrin-coated pits where it assists in membrane fission. Recently, however, this model has become controversial and an alternative proposal has emerged suggesting dynamin simply acts as a molecular switch similar to other GTPases. Additional dynamin family members have been implicated in numerous fundamental cellular processes, including other membrane fission events, anti-viral activity, cell plate formation and chloroplast biogenesis. Among these proteins, self-assembly and oligomerization into ordered structures (i.e. rings and spirals) is a common characteristic and, for the majority, essential for their function. While they are continually being implicated in diverse functions of the cell, we would like to know if a common mechanism of action exists. We are currently examining other dynamin family members including the MxA, a protein involved in fighting viral infection and Drp1, a dynamin related protein involved in mitochondria fission. Previously we demonstrated that dynamin undergoes a GTP-dependent conformational change causing constriction and fragmentation. Purified recombinant dynamin binds to lipid vesicles to form helical tubes. Treatment of these tubes with GTP causes a rapid alteration in structure, which ultimately leads to constriction and fragmentation. We believe this represents a critical step in the process that occurs when clathrin-coated pits bud from the plasma membrane. The ability of dynamin to constrict and generate a force on the underlying lipid bilayer makes it unique among GTPases as a mechanochemical enzyme. To further explore the dynamics of dynamin during GTP hydrolysis we have applied the novel technique of time-resolved cryo-electron microscopy. We observed that immediately upon GTP addition (within 5 seconds) dynamin constricts the underlying lipid bilayer in a concerted action and excess lipid bulges out at focal points along the constricted tubes. We are currently exploring different dynamin mutants, lipid, nucleotide and temperature conditions to mimic the in vivo environment. To determine the conformational changes that occur during dynamin-induced constriction we have calculated the first three-dimensional map of dynamin in the constricted state using cryo-electron microscopy and helical reconstruction methods at a resolution of 20 Angstroms. The map was determined using a dynamin mutant lacking the proline rich C-terminus (DPRD) in the presence of a GTP analogue. The 3D map consists of a repeating T structure (dimer) along the tube axis, which can be divided into three distinct densities called head, stalk and leg. Based on previous biochemical results and the docking of X-ray crystal structures into our map, we predict that the GTPase domain is located in the head and the PH domain is located in the leg. This leaves the middle domain and GTPase effector domain (GED) most likely located in the stalk. The positioning of GED within the stalk fits with previous findings that GED directly interacts in trans with a GTPase domain to stimulate the GTPase activity of dynamin. The constriction observed by GTP binding results from a decrease in both radial diameter and axial repeat. Based on our 3D map, an interaction between GED and a GTPase domain from a neighboring dimer could lead to both a radial and axial constriction.